Project Summary: Our understanding of the functions of T cell costimulatory pathways is undergoing an evolutionary process. It is now clear that in addition to provision of positive signals to promote T cell activation, T cell costimulatory pathways can provide negative/inhibitory signals that inhibit T cell activation to terminate immune responses and promote tolerance. In addition, the expression of costimulatory ligands on parenchymal cells has important functions in regulating immune responses at the level of the target organ. During the initial funding period of this RO1, we have generated novel data to show that the negative T cell costimulatory pathways play a critical role in physiologic regulation of alloimmune responses and tolerance. Our overall hypothesis is that the negative T cell costimulatory pathways regulate the physiologic (natural) and acquired tolerance to alloantigens at the level of both hematopoietic and parenchymal target organ cells. The main goal of this proposal is to define the functions and mechanisms of selected negative T cell costimulatory pathways in regulating alloimmune responses in vivo in experimental models of solid organ and tissue transplantation, as a means of achieving reproducible and durable in stringent transplant models to translate ultimately to experimental primates and humans. Our specific aims are: AIM I: To investigate the functions and mechanisms of the negative T cell costimulatory pathway PD-1:PDL1/PDL2 in alloimmune responses in vivo using models of solid organ and tissue (skin) transplantation. We will study the role of the PD-1 pathway in spontaneous engraftment and development of chronic allograft rejection focusing on parencyhymal expression of PD ligands in regulating alloimmunity. AIM 2: To investigate the role of the PD-1 pathway in acquired transplantation tolerance to allografts, and dissect the interactions with positive costimulatory pathways and role of Tregs. AIM 3: To investigate the functions and mechanisms of the negative T cell costimulatory pathway B7-H3 in alloimmune responses in vivo using models of solid organ and tissue (skin) transplantation. AIM 4: To investigate the functions and mechanisms of the negative T cell costimulatory pathway B7-H4 in alloimmune responses in vivo using models of solid organ and tissue (skin) transplantation. In order to define the exact functions of B7-H3 and B7-H4 pathways in alloimmunity and tolerance we also plan to clone and characterize the B7-H3 and B7-H4 receptors. This will be done in collaboration with Dr. Gordon Freeman. Lessons learned from this proposal will further our understanding of the mechanisms of allograft rejection and tolerance, and should lead to development of novel therapeutic strategies by harnessing the physiologic mechanisms that regulate immune responses and maintain tolerance. Narrative: Transplantation is now the treatment of choice for end stage organ failure. The major goal in transplantation is induction of a state of immunologic tolerance where the graft is permanently accepted without continuous toxic immunosuppression. The major goal of this proposal is to understand the physiologic mechanisms that regulate immune responses against transplanted organs and harness this knowledge to develop new therapies to ultimately induce transplantation tolerance in humans.